Halitosis, frequently associated with volatile sulfur compounds (VSCs) produced by oral microbiota, affects a large proportion of adults. Among VSCs, methyl mercaptan (CH₃SH) is a critical biomarker for periodontitis-related halitosis due to its strong correlation with periodontal pocket depth and attachment loss. This study investigated the utility of a methionine challenge protocol to selectively stimulate CH₃SH production and enhance the standardization of oral air-based screening for periodontal disease. Thirty adults were enrolled and divided equally into control and periodontitis groups. Mouth air samples were collected from oral cavity air using a straw-based sampling method connected to a portable gas-sensing device, which continuously monitored VSCs, including CH₃SH and hydrogen sulfide (H₂S), across eight time points. Participants underwent an 8 h fast prior to baseline oral air collection, followed by standardized toothbrushing. After a 60 min rest period, they swilled with a methionine solution, with oral air samples collected immediately after and at 10 min intervals for 40 min. Both groups showed increased CH₃SH levels following methionine stimulation, with the periodontitis group exhibiting a significantly greater increase from pre- to post-stimulation (p < 0.001) and higher cumulative exposure (p < 0.001). In contrast, H₂S levels remained consistently elevated in the periodontitis group but did not fluctuate significantly over time. Furthermore, correlations between CH₃SH and H₂S decreased immediately post-stimulation and gradually recovered in the periodontitis group. These findings indicate that the methionine challenge effectively induces CH₃SH production linked to periodontal dysbiosis, supporting its potential as a non-invasive screening and indicator tool for the presence of periodontitis, rather than for staging disease severity. The protocol offers a promising approach to improve diagnostic accuracy while minimizing variability related to oral hygiene. (The study is registered with the Clinical Research Information Service under number KCT0010328.).